Regulation of intracellular pH in LLC-PK 1 cells studied using 31P-NMR spectroscopy

Abstract

31P-NMR spectroscopy was used to monitor intracellular pH (pH i) in a suspension of LLC-PK 1 cells, a renal epithelial cell line. The regulation of intracellular pH (pH i) was studied during intracellular acidification with 20% CO 2 or intracellular alkalinization with 30 mM NH 4Cl. The steady-state pH i in bicarbonate-containing Ringer's solution (pH o 7.40) was 7.14 ± 0.04 and in bicarbonate-free Ringer's solution (pH o 7.40) 7.24 ± 0.04. When pH o was altered in nominally HCO − 3-free Ringer's, the intracellular pH i changed to only a small extent between pH o 6.6 and pH o 7.6; beyond this range pH i was linearly related to pH o. Below pH o 6.6 the cell was capable of maintaining a ΔpH of 0.2 pH unit (inside more alkaline), above pH 7.6 a ΔpH of 0.4 unit could be generated (inside more acid). During exposure to 20% CO 2 in HCO − 3-free Ringer's solution, pH i dropped initially to 6.9 ± 0.05, the rate of realkalinisation was found to be 0.071 pH unit·min −1. After removal of CO 2 the pH i increased by 0.65 and the rate of reacidification was 0.056 pH unit·min −1. Exposure to 30 mM NH 4Cl caused a raise of pH i by 0.48 pH unit and an initial rate of re-acidification of 0.063 pH unit·min −1, after removal of NH 4Cl the pH i fell by 0.58 pH unit below the steady-state pH i, followed by a subsequent re-alkalinization of 0.083 pH unit·min −1. Under both experimental conditions, the pH i recovery after an intracellular acidification, introduced by exposure to 20% CO 2 and by removal of NH + 4, was found to be inhibited by 53% and 63%, respectively, in the absence of sodium and 60% and 72%, respectively, by 1 mM amiloride. These studies indicate that 31P-NMR can be used to monitor steady-state intracellular pH as well a pH i transients in suspensions of epithelial cells. The results support the view that LLC-PK 1 cells use an Na +-H + exchange system to readjust their internal pH after acid loading of the cell.